Silencer sirna transfection

Expression of Runx1t1 in BV2 microglial cells was inhibited using specific siRNA (Silencer siRNA transfection, Cat No. 4390771, Ambion, Inc. USA). Pre-designed Runx1t1 siRNA and Scrambled siRNA were obtained from Ambion (Table 1). The siRNA transfection in microglial cells was performed according to the manufacturer’s instruction. BV2 microglial cells were resuspended in OPTIMEM medium at a concentration of 1×10⁵ cells/ml. Transfection complexes were prepared by mixing RNAiMAX Lipofectamine transfection agent (Cat No. 13778-150, Ambion) and siRNA (20 nM) in OPTIMEM Serum-free Medium (Invitrogen Life Technology, USA ). Microglial cells were then mixed with transfection complexes and incubated for 24 h in 6-well (2×10⁵ cells/well) or 24-well (4×10⁴ cells/well) plates.
Runx1t1 protein expression in microglial cells was analyzed by immunofluorescence (n = 3). Transfected BV2 microglial cells were fixed with 4% paraformaldehyde in phosphate buffer for 30 min at room temperature. Following incubation in normal goat serum, all coverslips with adherent cells were incubated in rabbit anti-Runx1t1 (1∶100, Cat.No.sc-28693, Santa Cruz, USA) at 4°C overnight. Subsequently, the cells were washed with PBS and incubated with FITC-conjugated lectin and Cy3-conjugated anti-rabbit IgG (1∶200, Sigma) for 1 h. Cells were finally counterstained with DAPI (1 µg/ml, Invitrogen, USA).